Acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) catalyzes the ATP-dependent carboxylation of acetyl-CoA to produce malonyl-CoA. This reaction occurs in two steps, carboxylation of a biotin prosthetic group using HCO.sup.-.sub.3 as a carboxyl donor, followed by a transfer of the carboxyl group from biotin to acetyl-CoA. ACCase in prokaryotes is composed of biotin carboxylase, biotin carboxyl carrier protein, and carboxyl-transferase alpha and beta subunits, each associated with different polypeptides. Samols, D. et al., J. Biol. Chem. 263:6461-6464 (1988). In contrast, ACCase of non-plant eukaryotes is comprised of multimers of a single multifunctional polypeptide. In plants, evidence of prokaryotic type ACCase (also known as the multi-subunit or heteromeric ACCase type) (Kannangara, C. G. et al., Arch. Biochem. Biophys. 152:83-91 (1972); Nikolau, B. J. et al., "The Biochemistry and Molecular Biology of Acetyl-CoA Carboxylase and Other Biotin Enzymes," In N. Murata, C. Somerville, eds., Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, American Society of Plant Physiologists, Rockville, Md. pp. 138-149 (1993) and Sasaki, Y. et al., J. Biol. Chem. 268:25118-25123 (1993)) has been obtained, and has been shown to be present in plastids of dicotyledons and of non-Gramineae monocotyledons (Konishi et al., 1996). A eukaryotic type (also known as the multi-functional or homomeric ACCase type) (Harwood, J. L., Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:101-138 (1988)) is probably present in the cytosol of all plant species.
The malonyl-CoA produced by ACCase is used in a wide variety of reactions and pathways in plants, including fatty acid synthesis and elongation (Harwood, J. L., Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:101-138 (1988)), flavonoid synthesis (Ebel, J. et al., Eur. J. Biochem. 75:201-209 (1977) and Ebel, J. et al., Arch. Biochem. Biophys. 232:240-248 (1984)), malonation of the ethylene precursor aminocyclopropane-1-carboxylate (Liu, Y. et al., Planta 158:437-441 (1983); Kionka, C. et al., Planta 162:226-235 (1984)) and malonation of amino acids and glycosides. Malonyl-CoA must be available in multiple subcellular locations, because some of these reactions, such as fatty acid synthesis, occur in the plastid while others, such as flavonoid synthesis and fatty acid elongation, occur outside the plastid. For example, very long chain fatty acids are components of plasma membrane lipids (Cahoon, E. B. et al., Plant Physiol. 95:58-68 (1991)) and are also needed for synthesis of cuticular waxes to cover the surface of both aerial and underground tissues. Harwood, J. L., Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:101-138 (1988). These very long chain fatty acids are synthesized outside the plastid by elongation of 16 or 18 carbon fatty acids exported from the plastid. Malonyl-CoA for the elongation reactions must be present in the cytosol, and is presumably provided by a cytosolic ACCase.
Malonyl-CoA must also be available in greatly differing amounts with respect to time and tissue. For example, increased amounts of malonyl-CoA are needed for fatty acid synthesis in developing seeds of species which store large quantities of triacylglycerols. Post-Beitenmiller, D. et al., "Regulation of Plant Lipid Biosynthesis: An Example of Developmental Regulation Superimposed on a Ubiquitous Pathway," In DPS Verma, ed., Control of Plant Gene Expression, CRC press, Boca Raton, Fla. pp. 157-174 (1993). In floral tissue, malonyl-CoA is used in the chalcone synthase reaction for synthesis of the flavonoid pigments which constitute up to 15% of the dry weight of this tissue. Goodwin, T. W. et al., "Introduction to Plant Biochemistry," 2nd ed., Pergamon Press New York, p. 545 (1983). In some tissues, ACCase might provide malonyl-CoA constitutively to produce fatty acids for membrane synthesis and maintenance, while providing a "burst" of malonyl-CoA for only a short period to synthesize flavonoids during exposure to UV light (Ebel, J. et al., Eur. J. Biochem. 75:201-209 (1977)) or during fungal pathogen attack. Ebel, J. et al., Arch. Biochem. Biophys. 232:240-248 (1984).
The possible roles of both ACCase, and another enzyme, 3-ketoacyl-ACP synthase III (KAS III), in plant fatty acid synthesis have been examined. KAS III has been suggested as an enzyme that limits fatty acid synthesis and the oil content of oilseed crops. An E. coli KAS III gene has now been overexpressed in transgenic rapeseed, resulting in 3 to 4 fold higher KAS III activity. Verwoert, IIGS et al., Plant Mol. Biol. 26(1):189-202 (1994). Although fatty acid composition was altered, indicating in vivo activity of the E. coli enzyme, total seed fatty acid content was not significantly changed.
While ACCase has not been previously overexpressed in plants, considerable evidence suggests that this enzyme is involved in regulation of plant fatty acid synthesis, and various observations have also led to the belief that ACCase may be the rate-limiting enzyme for oilseed fatty acid synthesis. Analysis of substrate and product pool sizes has implicated ACCase in the light/dark regulation of fatty acid synthesis in spinach leaves and chloroplasts. Post-Beitenmiller, D. et al., J. Biol. Chem. 266:1858-1865 (1991) and Post-Beitenmiller, D. et al., Plant Physiol. 100:923-930 (1992). ACCase may also be the site of feedback inhibition of fatty acid synthesis in tobacco suspension cells supplemented with exogenous fatty acids. Shintani, D. K. et al., Plant Physiol. 102:S-11 (1993). Furthermore, ACCase activity increases in association with lipid deposition in developing seeds of oilseed crops. Simcox, P. D. et al., Canada J. Bot. 57:1008-1014 (1979); Turnham, E. et al., Biochem. J. 212:223-229 (1983); Charles et al., Phytochem. 25:55-59 (1986) and Deerburg, S. et al., Planta 180:440-444 (1990). ACCase therefore appears to have a very important regulatory role in plant fatty acid synthesis.
It would thus be desirable to provide a gene encoding acetyl-CoA carboxylase (ACCase). It would also be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA. It would further be desirable to control the carboxylation of acetyl-CoA to produce malonyl-CoA by controlling the expression of a gene encoding ACCase. It would further be desirable to acquire long-term control of the carboxylation of acetyl-CoA to produce malonyl-CoA by genetically altering plants. It would also be desirable to control fatty acid synthesis and elongation in plants and seeds by controlling the expression of a gene encoding ACCase. It would further be desirable to control fatty acid synthesis and elongation in plants and seeds without employing foreign chemicals. It would also be desirable to control the production of plant secondary metabolites.